Identification of Rbfox-regulated transcripts important for muscle function
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Series/Report no.:Ohio State University. Undergraduate Research Festival. 2018 Autumn. 3-Minute Thesis Competition.
Rbfox RNA-binding proteins are important regulators of muscle-specific splicing. Rbfox proteins contain an RNA recognition motif that is conserved from flies to human and binds to (U)GCAUG motifs that are enriched in introns next to muscle-specific alternative exons. Many muscle-specific splicing events are conserved among vertebrates, and we use the zebrafish model to understand how Rbfox-mediated splicing regulates muscle development and function. In zebrafish, Rbfox1l and Rbfox2 are both expressed in muscle. Double knockdown of rbfox1l and rbfox2 using antisense morpholinos leads to splicing changes of muscle-specific alternative exons, coupled with skeletal muscle paralysis and reduced heart rate. To confirm whether splicing changes and phenotypic effects in morphants are indeed due to Rbfox depletion and not morpholino-induced side effects, I compared phenotype and alternative splicing changes in rbfox genetic mutants versus morphants. Using our unpublished RNA-seq data, I identified candidate Rbfox-regulated transcripts and focused on transcripts that are highly expressed and show substantial splicing changes between wildtype and double morphant embryos. To enrich for direct targets, I selected only those candidates containing intronic Rbfox motifs for further analysis. 9 of 12 (75%) tested candidates were similarly affected in double mutants and double morphants. These results indicate that splicing is similarly affected in morphants and mutants, consistent with the observation that rbfox double mutants and double morphants also have the same morphological phenotypes. Future experiments will test whether Rbfox-dependent transcript isoforms can fully or partially rescue muscle function when microinjected into double mutants. Transcript isoforms that restore muscle function in mutants are likely targets of Rbfox regulation that are necessary for proper muscle development. Our long-term goal is to identify Rbfox-regulated splicing events that are required for muscle function. The insight gained from this work will improve our understanding of Rbfox-regulated muscle splicing and may provide clues for the development of therapies for human muscle diseases.
Academic Major: Molecular Genetics